A large number of nitrile hydratases have already been described in the literature (Synthetic applications of nitrile-converting enzymes; Martinkova, Ludmila; Mylerova, Veronika; Current Organic Chemistry (2003), 7(13), 1279-1295). Nitrile hydratases have been used since 1983 for producing acrylamide on a scale of several thousand tons per year. This biocatalytic process has proved to be able to compete with the chemical processes (Enzymic synthesis of acrylamide: a success story not yet over; Kobayashi, Michihiko; Nagasawa, Toru; Yamada, Trends in Biotechnology (1992), 10(11), 402-8).
In addition to the nitrile hydratases which can be used for converting acrylonitrile, nitrile hydratases which are are particularly suitable for converting methacrylonitrile (A nitrile hydratase of Pseudonocardia thermophila and the genes encoding and manufacture of the enzyme for conversion of nitriles to amides (EP 790310), 3-cyanopyridine (Process for producing amides with Rhodococcus nitrile hydratase (WO 2002055670) or 2-hydroxynitriles such as 2-hydroxy-4-methylthiobutyro-nitrile (A nitrile hydratase of Rhodococcus and its use in the manufacture of amides (WO 2002070717) and Enzymic conversion of α-hydroxynitriles to the corresponding α-hydroxyamides, acids or acid salts, (WO 9832872) have, for example, also been described. By contrast, no nitrile hydratases which can be used to efficiently convert 2-aminonitriles are thus far known. While the Rhodococcus sp. Cr4 nitrile hydratase converts 2-hydroxynitriles, for example, with a high degree of activity, it does not convert a simple 2-aminonitrile such as aminoacetonitrile at all (WO 2002070717).
The enzymic conversion of aminonitriles into the corresponding amides opens up an attractive route for synthesizing amino acids since 2-aminoamides can be hydrolyzed readily (WO 2001060789). This process proceeds under mild conditions and with a very high degree of selectivity and without the formation of byproducts such as salts, as accrue in connection with chemical hydrolysis.
Alternatively, amides can also be converted with alkali metal or alkaline earth metal hydroxides into the corresponding salts of the acids. This approach is particularly preferred when using calcium hydroxide for converting 4-methylthio-α-hydroxybutyramide (MHA-amide), since the calcium salt of MHA can be used directly as a feedstuff additive, as a product form which is an alternative to methionine or MHA.
However, for producing a commodity product such as DL-methionine, it is not sufficient to make available a high-activity biocatalyst. In order to increase the activity, it is necessary to establish a system for expressing the genes which are to be amplified. One possibility which presents itself is heterologous expression, for example, and in particular, in Escherichia coli, Bacillus, Pseudomonas, Pichia, Sacharomyces or Aspergillus, since these microorganisms exhibit rapid growth, achieve very high cell densities and are available molecular biological tools which permit very high expression levels (Lee S Y (1996) High cell-density culture of Escherichia coli. TIBTECH 14:98-105; Riesenberg D, Guthke R (1999) High-cell-density cultivation of microorganisms. Appl Microbiol Biotechnol 51:422-430).
It is known that at least 3 genes have to be coexpressed for nitrile hydratases to be expressed heterologously. In addition to two structural genes, a corresponding auxiliary protein has to be amplified both for iron-dependent and for cobalt-dependent enzymes (Nojiri M. et al., (1999) Functional expression of Nitrile hydratases in Escherichia coli: Requirement of a nitrile hydratase activator and a post-translational modification of a ligand cysteine. J Biochem 125: 696-704 and Over-production of stereoselective nitrile hydratase from Pseudomonas putida 5B in Escherichia coli: activity requires a novel downstream protein, Wu, S.; Fallon, R. D.; Payne, M. S. Applied Microbiology and Biotechnology (1997), 48(6), 704-708).
In addition to these 3 genes, a further gene, which encodes a cobalt transporter, was found, alongside the structural genes and the auxiliary protein gene, in a gene cluster in Rhodococcus rhodochrous J1 (A novel transporter involved in cobalt uptake, Komeda, Hidenobu et al., Proceedings of the National Academy of Sciences of the United States of America (1997), 94(1), 36-41). Overexpression in both Rhodococcus and in E. coli leads to an increased uptake of Co2+ ions from the culture medium. In addition, it was shown that, when the cobalt transporter is coexpressed together with the 3 other proteins, it is possible to achieve the same nitrile hydratase activity at a concentration of Co in the medium which is lower than when the structural genes and the auxiliary protein are expressed on their own. However, according to Komeda et al., this effect only occurs in Rhodococcus at concentrations of less than 42 μM.
EP 0 362 829 discloses the fermentation of Rhodococcus rhodochrous in the presence of cobalt salts.